耐高温β-半乳糖苷酶突变体的真核表达、高效制备及特性分析

发布时间：2018-04-05 23:14

本文选题：β-半乳糖苷酶　切入点：酶活力　出处：《山东大学》2017年硕士论文

【摘要】：β-半乳糖苷酶(β-galactosidase,EC3.2.1.23),简称乳糖酶(lactase),普遍存在于动物组织、植物及微生物中,具有将乳糖分解为半乳糖和葡萄糖的能力,并能够催化产生半乳糖寡聚体,即低聚半乳糖(Galactooligosaccharides,GOS)。目前在食品工业和市场上,乳糖酶的这两个酶反应特性具有不同的实用意义。乳糖不耐症是一个全球普遍存在的生理性问题,并且人们因为诸多原因都有可能从非乳糖不耐症的群体发展成为乳糖不耐症患者,因此无/低乳糖乳制品一直以来都受到特别的关注。另一方面,近年来国内生活质量的飞升,引起了多种相关疾病(肥胖、三高等)高发,这些病种在国内有上升趋势,因此人们对膳食管理和食品安全倍加重视。研究表明GOS具有平衡肠道菌群,调节肠道酸碱度,预防便秘,改善人体肠道的生理功能,辅助钙质吸收,加速脂肪代谢,保护肝脏功能等多种特质,这使得GOS这种新型的天然食品添加剂有了广阔的市场空间。而这些方向都是乳糖酶可能涉及的市场和工业领域。国内对于乳糖酶耐高温性质和乳糖酶水解方向的研究较多,但对于GOS合成方向的研究刚刚起步。这些研究中,活性中心单个位点的氨基酸对整个乳糖酶双向性质影响的研究很少。国际上研究也比较少,只有少数科学家在做这方面相关的研究。因此在基于位点变化而影响乳糖酶分解能力或合成能力的研究上还有很大的空间。本研究通过点突变使β-半乳糖苷酶[Pyrococcus furiosus DSM 3638]415位天冬酰胺(Asn or N)突变为丝氨酸(Ser or S),突变体构建于巴斯德毕赤酵母表达质粒载体,通过转基因的方法把此线性化的重组质粒整合入巴斯德毕赤酵母细胞中,经药物筛选获取单克隆的细胞株,用以发酵表达进而制备β-半乳糖苷酶的突变体酶。研究结果表明,该突变体所产的β-半乳糖苷酶在95℃、pH=5.5时酶活力达到最高,为耐高温乳糖酶;该乳糖酶的热稳定性良好,100℃加热1h条件下,几乎不影响该乳糖酶的活性;该突变体酶在低温储存环境下活性也十分稳定;研究表明,该突变体酶水解牛奶的能力较好,可用于低乳糖牛奶及其相关制品加工中;突变后β-半乳糖苷酶的另一个方面的特性,即产生低聚半乳糖的能力的也有所提高,但不够明显,低于可工业化的水平。因此该突变株所产的乳糖酶可用于乳制品的加工行业,但不能期盼产生更多的半乳糖寡聚体。同时研究通过中试级别乳糖酶生产发现该乳糖酶的表达活力随菌体密度的增加而上升,这对于该菌株用于工业生产乳糖酶及后期纯化十分有利。由研究可推测:源自Pyrococcus furiosus DSM 3638的乳糖酶基因耐热程度较好。同时,位点氨基酸的改变影响了活性中心的构象,从而在酶的稳定性上得到了较大的提升。该突变位点所在活性中心可能更多的作用于水解乳糖的过程,但是对于GOS的合成并没有太大的关联。由此可进一步猜测,或许该耐热乳糖酶合成GOS的能力另受其他位点的影响。
[Abstract]:Beta galactosidase (beta -galactosidase, EC3.2.1.23), (lactase), commonly referred to as lactase exists in animal tissues, plants and microorganisms, has ability to dissolve lactose galactose and glucose, and can produce galactose oligomers, i.e. galactooligosaccharides (Galactooligosaccharides, GOS). At present in food industry and market, has different practical significance of the two lactase enzyme reaction characteristics. Lactose intolerance is a universal psychological issue, and people for many reasons are likely from non lactose intolerance group become lactose intolerant, so no / low lactose dairy products have always attracted special attention. On the other hand, the quality of life of domestic soaring in recent years, due to a variety of related diseases (obesity, high incidence of these diseases, three higher) is on the rise in China, so people Pay attention to food management and food safety. More studies showed that GOS has the balance of intestinal flora, regulating intestinal pH, prevent constipation, improve the physiological function of the human intestinal tract, assisted calcium absorption, accelerate fat metabolism, protect liver function and other characteristics, which makes GOS the new natural food additive has a broad market space which direction is lactase may be involved in the market and industry. There are many researches on high temperature properties of lactase and lactase hydrolysis direction, but the study of GOS synthesis has just started. These studies, little effect of amino acid on the activity of a single center site. The two-way nature of lactase research relatively small, only a few scientists have done related research in this area. Based on site changes affect the lactase synthesis ability or capacity There is a lot of space research. The study of the beta galactosidase [Pyrococcus furiosus DSM 3638]415 (Asn or point mutation by asparagine to serine mutation (Ser N) or S), the mutant constructed Pasteur Pichia expression vector, through the method of transgenic recombinant plasmid to this linear integrated into Pasteur Pichia pastoris, the drug screening to obtain monoclonal cell lines with mutant enzyme preparation and expression of beta galactosidase in fermentation. The results showed that the mutants produced by beta galactosidase at 95 degrees, pH=5.5 enzyme activity reached the highest, the high-temperature thermal stability of the lactase; good 100 degrees of lactase, heating under the condition of 1H, almost does not affect the lactase activity; the mutant in the environment of low temperature storage activity is stable; research shows that the ability of the mutant enzyme hydrolysis of milk Well, can be used for low lactose milk and related products; on the other hand the characteristics of beta galactosidase mutation, produce oligomate ability is also improved, but obviously not enough, below the level of industrialization. Therefore the mutant strain produced by lactose enzyme can be used for dairy processing industry products, but cannot be expected to produce more galactose oligomers. At the same time through the research on the test level of lactase production showed that the expression of the lactase activity increased with increasing cell density, which for the strains used in industrial production and purification of lactase after period is very favorable. From the research that: from the good heat level of lactase gene Pyrococcus furiosus DSM 3638. At the same time, amino acid changes affecting the conformation of active center, thus greatly enhance the stability of the enzyme. The mutation site activity The heart may play more role in the hydrolysis of lactose, but it has little relationship with the synthesis of GOS. Further speculation is made that the ability of the thermostable lactase to synthesize GOS is also affected by other sites.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55;Q78

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